Influenza A and B viruses are both members of the genus Influenzavirus of the Family Orthomyxoviridae, and as such are closely related in genome, structure, and pathogenicity. Research directed at influenza B virus has benefited greatly from studies performed on influenza A viruses. While many points of similarity exist, differences ar emerging at the molecular genetic level between these two virus types. These differences can be important in the treatment of influenza, e.g. type B influenza is resistant to the antiviral activity of amantadine and its derivatives while type A influenza is not. Our long range goal is to illuminate these differences as they exist in influenza B virus, and use this information to help control this virus. Our specific aims are geared to accomplish two tasks. The first goal is to establish a large, comprehensive set of influenza type B ts mutant viruses, each with one mutation in one gene. This set of mutants is a basic resource needed for future work in influenza B virus genetics, and, at present, is nonexistent. The second goal is to utilize this resource to explore the mystery of the matrix gene's apparently unutilized but conserved second reading frame (orf). RNA segment 7, the matrix (M) gene of influenza B virus has been shown to contain two orfs analogous to the orfs of RNA 7 in influenza A viruses which encode M1 and M2 proteins, but, as yet, no M2 mRNA and no M2 protein have been found. In line with this lack of M2 protein, influenza B viruses are insensitive to the action of amantadine derivatives at the low levels active against the M2 protein of influenza type A viruses. Still, in all M genes of influenza B viruses sequenced to date, this second orf has been conserved. It is highly unlikely that this conservation over time would occur in a highly mutable virus as influenza, without the selection pressure of a protein product that is essential for a productive virus infection. Since conventional approaches that have tried isolating M2 mRNA and/or M2 protein have been unsuccessful, we will attempt to verify the necessity for this second orf by isolating ts mutants caused by lesion(s) within its coding region. Our set of ts mutants will be screened for mutations within the M2 region by mapping the existence and location of point mutations with the RNase mismatch cleavage method, followed by subsequent linkage of this gene to the ts phenotype by genetic reactivation experiments. The existence of ts mutants in this area will establish a functional requirement for the second orf, and should also help in the subsequent search and unambiguous identification of the M2 gene product and greatly aid in discovering its function.